CN114392357B - Cell membrane anchored nucleic acid medicine, preparation method and application thereof - Google Patents
Cell membrane anchored nucleic acid medicine, preparation method and application thereof Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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Abstract
The invention discloses a cell membrane anchored nucleic acid drug, a preparation method and application thereof. The nucleic acid agent comprises a first anchor nucleic acid sequence and a regulatory nucleic acid sequence; the regulatory nucleic acid sequence comprises a nucleic acid aptamer sequence and a second anchor nucleic acid sequence; the 5' end of the nucleic acid aptamer sequence is connected with a second anchoring nucleic acid sequence, the second anchoring nucleic acid sequence and the first anchoring nucleic acid sequence are completely complementary and hybridized to form a cell membrane anchoring sequence with a double-chain structure, and one or two cell membrane anchoring groups are modified by the cell membrane anchoring sequence. The preparation method of the nucleic acid aptamer comprises the following steps: s1, synthesizing a first anchor nucleic acid sequence; s2, synthesizing a regulatory nucleic acid sequence; s3, mixing the two nucleic acid sequences according to the molar concentration of 1:1, and heating at 95 ℃ for 5 minutes to cool. The nucleic acid medicine can improve the combination stability of the nucleic acid aptamer medicine and the target receptor protein, prolong the acting time on the cell membrane and improve the regulation and control effect of the nucleic acid aptamer medicine on the cell membrane receptor protein.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a cell membrane anchored nucleic acid drug, a preparation method and application thereof.
Background
The receptor proteins on the surface of the cell membrane are important signal proteins for maintaining the normal physiological functions of cells. Abnormal expression or dysfunction of these receptor proteins is closely related to many disease processes including cancer, diabetes, neurodegenerative diseases, and the like. In recent years, many studies have been conducted to develop various monoclonal antibodies or small molecule inhibitors, etc. with cell membrane receptor proteins as drug targets, to achieve inhibition of protein activity. However, the production of these formulations is time consuming, labor intensive and costly, difficult to control and prone to immune responses, limiting further use. In recent years, due to the advantages of easy synthesis and modification, controllable quality, easy storage and low immunogenicity, aptamer medicines are gradually exposed in the research of developing nucleic acid medicines with disease treatment functions. Some specific nucleic acid aptamers are reported to be useful as antitumor agents directly, exhibiting potential for inhibiting the activity of a target protein by preventing its interaction with other signaling proteins, such as binding of a receptor to a ligand. Although aptamer drugs can play a therapeutic role, there are some limitations. In a first aspect, the aptamer drug typically employs self-steric hindrance after binding to the target to inhibit the original receptor-ligand interaction mediated protein activation process. In complex cellular microenvironments, free aptamer drugs are susceptible to dissociation after binding to the target molecule due to their small size, resulting in generally large doses of aptamer drugs being required to exert inhibitory functions. In the second aspect, due to endocytosis of cells, the nucleic acid aptamer medicine taking a cell membrane receptor protein as a target cannot continuously exert an inhibition function after being endocytosed by tumor cells, so that the inhibition time of the nucleic acid aptamer medicine is short. Therefore, the combination stability of the nucleic acid aptamer medicine is improved, the residence time of the nucleic acid aptamer medicine on a cell membrane is prolonged, the inhibition effect of the nucleic acid aptamer medicine can be improved, and the application prospect of preparing the nucleic acid medicine for treating diseases is wider.
In order to solve the above technical problems, there is a need to develop a stable and efficient cell membrane anchored nucleic acid drug, a preparation method and application thereof.
The invention comprises the following steps:
the invention aims to provide a cell membrane anchored nucleic acid drug, a preparation method and application thereof, and the technical problems to be solved include but are not limited to any one of the following technical problems: in a first aspect, how to improve the binding stability of a nucleic acid aptamer drug to a target receptor protein; in a second aspect, how to realize the efficient inhibition of cell membrane receptor protein activity by the nucleic acid aptamer medicament, and reduce the dosage of the nucleic acid aptamer medicament; in a third aspect, how to achieve long-acting inhibition of cell membrane receptor protein activity and the like of a nucleic acid aptamer drug.
In order to solve any one or more of the technical problems, the invention adopts the following technical scheme:
Providing a cell membrane anchored nucleic acid drug formed by the assembly of a first anchoring nucleic acid sequence and a regulatory nucleic acid sequence comprising two functional sequences: a nucleic acid aptamer sequence and a second anchor nucleic acid sequence; the 5' end of the nucleic acid aptamer sequence is connected with the second anchoring nucleic acid sequence, the nucleic acid aptamer sequence specifically targets cell membrane receptor proteins, the nucleic acid aptamer sequence is shown in SEQ ID NO.1, the second anchoring nucleic acid sequence and the first anchoring nucleic acid sequence are completely complementary and hybridized to form a cell membrane anchoring sequence with a double-chain structure, and one or two cell membrane anchoring groups are modified on the cell membrane anchoring sequence. In this embodiment, a cell membrane anchoring group is modified at the end or other position of the second anchoring nucleic acid sequence or the first anchoring nucleic acid sequence; two cell membrane anchoring groups are modified at the ends or other positions of the second anchoring nucleic acid sequence and the first anchoring nucleic acid sequence, respectively.
In another modified embodiment, 1 to 5 random base sequences are provided between the nucleic acid aptamer sequence and the second anchor nucleic acid sequence. By arranging 1-5 random base sequences between the nucleic acid aptamer sequence and other sequences, the purpose is to separate two functional sequences, keep the configuration of the nucleic acid aptamer sequence and not influence the nucleic acid aptamer sequence to play a role.
In another refinement of the above, the cell membrane anchoring group is one of hydrophobic molecules including cholesterol molecules, tocopherol molecules, and diacyl liposomes.
In a further modified embodiment, the first anchor nucleic acid sequence and the second anchor nucleic acid sequence are complementary hybridized to form a double-stranded structure having a length of 18-24 bp.
In a further development of the above-described variant, both of the cell membrane anchoring groups are modified in the middle of the first and the second anchoring nucleic acid sequences; the intermediate position means: the first anchoring nucleic acid sequence and the second anchoring nucleic acid sequence are n bases, and when n is even, the two cell membrane anchoring groups are respectively modified between the n/2 th and (n/2) +1 th bases of the first anchoring nucleic acid sequence (direction 5 '-3') and the second anchoring nucleic acid sequence (direction 3 '-5'); when n is an odd number, two of the cell membrane anchoring groups are modified in the middle of the (n/2) -0.5 th and (n/2) +0.5 th bases of the first anchoring nucleic acid sequence (direction 5 '-3') and the second anchoring nucleic acid sequence (direction 3 '-5'), respectively. This arrangement can further enhance the stability of the anchoring of the cell membrane anchoring sequence to the cell membrane.
In a further development of the above-described embodiment, the cell membrane receptor protein is a mesenchymal epidermal transforming factor (c-Met receptor protein) which is activated by receptor dimerization mediated by its ligand Hepatocyte Growth Factor (HGF).
In another modified embodiment, the binding site of the aptamer to the cell membrane receptor protein and the binding site of the ligand to the cell membrane receptor protein overlap or are identical.
The invention also provides a preparation method of the cell membrane anchored nucleic acid medicament, which comprises the following steps of,
S1, synthesizing a first anchor nucleic acid sequence;
s2, synthesizing a regulatory nucleic acid sequence;
s3, mixing the two nucleic acid sequences in the steps S1 to S2 according to the molar concentration of 1:1, heating at 95 ℃ for 5 minutes for annealing and hybridizing, and slowly cooling to room temperature.
The invention also provides application of the cell membrane anchored nucleic acid medicine in nucleic acid aptamer medicine.
The invention also provides application of the cell membrane anchored nucleic acid medicine in the research related to the regulation of the activity of c-Met receptor protein and the cell function.
The technical scheme of the invention has at least the following beneficial effects: the invention provides a cell membrane anchored nucleic acid drug, a preparation method and application thereof. The nucleic acid drug anchored by the cell membrane can obviously improve the combination stability of the nucleic acid aptamer drug and the target protein. The cell membrane anchored nucleic acid medicine of the present invention may be used in regulating cell membrane receptor protein activity and cell function.
(1) Some nucleic acid aptamers can be directly used as antitumor drugs, and interaction between target proteins and other signal proteins, such as binding of a receptor and a ligand, is prevented by utilizing self steric hindrance after the target is bound, so that the application potential of inhibiting the activity of the target proteins is shown. However, in complex cellular microenvironments, free aptamer is susceptible to dissociation after binding to the target molecule due to its small size, resulting in the need for a large dose of aptamer to exert an inhibitory function. Compared with free nucleic acid aptamer, the nucleic acid aptamer disclosed by the invention has the advantages that more nucleic acid aptamer is enriched on the surface of a cell membrane through the cell membrane anchoring sequence with a double-chain structure, and meanwhile, the binding stability is increased, so that the activity of c-Met receptor protein can be obviously inhibited at a lower concentration, and the inhibition effect of the nucleic acid aptamer is enhanced.
(2) Because of endocytosis of cells, the nucleic acid aptamer medicine taking cell membrane receptor protein as a target point cannot continuously exert the inhibition function after being endocytosed by tumor cells, so that the inhibition time of the nucleic acid aptamer medicine is short. The nucleic acid medicine of the invention utilizes the cell membrane anchoring sequence to slow down the endocytosis process of the nucleic acid aptamer sequence, and aims at targets on cell membranes such as c-Met receptor proteins, thereby prolonging the time of inhibition. Namely, the invention can effectively prolong the acting time of the aptamer on the surface of the cell membrane and reduce the loss of drug effect caused by endocytosis. Therefore, the cell membrane anchored nucleic acid medicine provides a new platform for developing high-efficiency nucleic acid medicines, and further expands the application prospect of the nucleic acid medicines in biomedicine.
Drawings
FIG. 1 is a schematic diagram showing the enhancement of inhibition of the activity of a target receptor protein by a cell membrane anchored nucleic acid drug of examples 1 and 2 of the present invention;
FIG. 2 shows confocal images of HeLa cells treated with different cell membrane anchoring sequences in a medium containing 10% FBS, (F) quantitative analysis of fluorescence intensity of each group of cells in examples 1 and 2 of the present invention; ruler: 20 μm;
FIG. 3 is a gel electrophoresis image of the first anchor nucleic acid sequence and the regulatory nucleic acid sequence of examples 1 and 2 of the present invention assembled;
FIG. 4 is a confocal image of 200nM 2CH-ab:b or 2CH-ad:d of example 1 and example 2 of the invention incubated with HeLa cells for 10 min at room temperature; ruler: 20 μm;
FIG. 5 is a confocal image of the anchoring directionality of 2CH-ab:b on cell membrane in examples 1 and 2 of the present invention; ruler: 20 μm;
FIG. 6 is a confocal image of the binding stability of comparative 2CH-ab: b and free a to c-Met receptor proteins in examples 1 and 2 of the present invention. Ruler: 20 μm;
FIG. 7 shows the effect of immunoblotting analysis on the control of the activity of the c-Met receptor protein in comparative examples 1 and 1 of examples 2CH-ab: b and ab: b;
FIG. 8 shows the effect of immunoblotting analysis of the activity of free a on c-Met receptor protein in comparative example 1 and comparative example 2 according to the present invention;
FIG. 9 immunoblot analysis of example 2 and comparative example 2 of the present invention compares the regulatory effect of 2CH-ad: d and ad: d on c-Met receptor protein activity;
FIG. 10 is a graph showing the migration ability of DU145 cells under various conditions, scale bar, of the cell scratch test of example 1 and example 2 of the present invention: 100 μm;
FIG. 11 shows the effect of immunoblot analysis comparing 2CH-ab: b and ab: b on c-Met receptor protein activity after 24 hours incubation with DU145 cells in examples 1 and 2 of the present invention.
Detailed Description
Hereinafter, specific examples of the present invention will be described in more detail using examples and comparative examples, and the technical scope of the present invention is not limited to the following examples.
Referring to the schematic of FIG. 1, the cell membrane anchored nucleic acid agent of the present invention is assembled from a first anchor nucleic acid sequence and a regulatory nucleic acid sequence comprising two functional sequences: a nucleic acid aptamer sequence and a second anchor nucleic acid sequence; wherein, the nucleic acid aptamer targets the cell membrane receptor protein, the nucleic acid aptamer sequence is shown in SEQ ID NO.1, the second anchor nucleic acid sequence and the first anchor nucleic acid sequence are completely complementary hybridized to form a cell membrane anchor sequence with a double-chain structure, and cell membrane anchor groups are modified on the second anchor nucleic acid sequence and the first anchor nucleic acid sequence.
In this example, 1 random base sequence is placed between the aptamer sequence and the second anchor nucleic acid sequence. This does not affect the ligation of the nucleic acid aptamer sequence to the second anchor nucleic acid sequence, while separating the two and does not affect the configuration of the nucleic acid aptamer sequence. In other embodiments, an integer number of random base sequences of 5 or less may be provided. However, the number of random base sequences in this scheme is not too large, the length of the nucleic acid aptamer sequence is 50 bases, the random base sequences are used for ensuring that the configuration of the nucleic acid aptamer sequence is not affected, if the number is too large, on the one hand, DNA is wasted, and on the other hand, the overlong random base sequences may affect the configuration of the nucleic acid aptamer sequence.
Based on the above embodiment, in another modified technical embodiment, the cell membrane anchoring group is one of hydrophobic molecules, the hydrophobic molecules are cholesterol molecules, the cholesterol molecules are modified on phosphate groups of the DNA backbone, and the hydroxyl groups and the phosphate groups after deprotection on the cholesterol molecules react to form ester bonds. In other embodiments, the hydrophobic molecule is a tocopherol molecule or a diacyl liposome. Since cholesterol molecules, tocopherol molecules and diacyl liposomes are all commonly used cell membrane anchoring groups and can be modified in DNA sequences, all three molecules anchor the DNA sequence to the cell membrane. In the experimental part of the present specification, cholesterol molecules are used as cell membrane anchoring groups, and it is known from the common knowledge in the art that the use of tocopherol molecules or diacyl liposomes instead of cholesterol molecules can also achieve corresponding experimental results.
In a further modified technical embodiment, based on the above embodiment, the first anchor nucleic acid sequence and the second anchor nucleic acid sequence complementarily hybridize to form a double-stranded structure having a length of 18-24 bp. The complete complementary hybridization of the second anchor nucleic acid sequence with the first anchor nucleic acid sequence results in a double-stranded cell membrane anchor sequence, which if too long in length, increases the difficulty of modifying the hydrophobic molecule and wastes DNA material and increases costs.
On the basis of the above embodiments, in a further improved technical embodiment, the cell membrane anchoring groups are each modified at a position intermediate the first anchoring nucleic acid sequence and the second anchoring nucleic acid sequence; the intermediate position refers to: the first anchoring nucleic acid sequence and the second anchoring nucleic acid sequence are n bases, when n is even, the two cell membrane anchoring groups are respectively modified between the n/2 th and (n/2) +1 th bases of the first anchoring nucleic acid sequence (with the direction of 5 '-3') and the second anchoring nucleic acid sequence (with the direction of 3 '-5'); when n is an odd number, the two cell membrane anchoring groups are modified in the middle of the (n/2) -0.5 th and (n/2) +0.5 th bases of the first anchoring nucleic acid sequence (direction 5 '-3') and the second anchoring nucleic acid sequence (direction 3 '-5'), respectively. This arrangement can further enhance the stability of the anchoring of the cell membrane anchoring sequence to the cell membrane.
Based on the above embodiments, in another modified technical embodiment, the cell membrane receptor protein is a mesenchymal epidermal transforming factor (c-Met receptor protein) capable of being activated by receptor dimerization mediated by its ligand Hepatocyte Growth Factor (HGF). The positions of the aptamer sequences in this example where the aptamer sequences bind to the c-Met receptor protein overlap the positions of the ligand HGF binding to the c-Met receptor protein, thus forming a competitive relationship. That is, after the aptamer sequence binds to the c-Met receptor protein, HGF is not easily bound to the c-Met receptor protein. The free aptamer sequence is easy to dissociate after being combined with the c-Met protein, and endocytosis and the like occur, so that the HGF can be partially combined with the c-Met receptor protein to activate the c-Met receptor protein. By attaching a double-stranded cell membrane anchoring sequence to the nucleic acid aptamer sequence, more nucleic acid aptamer is enriched on the cell membrane surface, the anchoring effect increases the binding stability, and the inhibition effect of the nucleic acid aptamer sequence becomes better. Therefore, the activity of c-Met receptor protein can be obviously inhibited at a lower concentration, and the inhibition effect of the nucleic acid aptamer medicine is enhanced; meanwhile, the cell membrane anchoring sequence is utilized to slow down the endocytosis process of the nucleic acid aptamer sequence, and the action time of the nucleic acid aptamer sequence on the cell membrane surface is effectively prolonged aiming at targets on the cell membrane such as c-Met receptor protein, so that the loss of drug effect caused by endocytosis is reduced.
Based on the above embodiments, in another modified technical embodiment, the binding site of the aptamer to the cell membrane receptor protein and the binding site of the ligand to the cell membrane receptor protein are partially overlapped or identical.
The invention also provides a preparation method of the nucleic acid medicament, which comprises the following specific steps,
S1, synthesizing a first anchor nucleic acid sequence;
s2, synthesizing a regulatory nucleic acid sequence;
S3, mixing the two DNA sequences in the steps S1 to S2 according to the molar concentration of 1:1, heating at 95 ℃ for 5 minutes for annealing and hybridizing, and slowly cooling to room temperature.
The invention also provides application of the nucleic acid medicine in nucleic acid aptamer medicines.
The invention also provides application of the nucleic acid medicine in the research related to the regulation of the activity of c-Met receptor protein and cell functions.
According to the invention, hydrophobic cell membrane anchoring groups are modified at the middle positions of the first anchoring nucleic acid sequence and the second anchoring nucleic acid sequence, so that the assembled nucleic acid aptamer can be stably anchored on the surface of a cell membrane. When the cell membrane-anchored nucleic acid agent of the present invention is used as a nucleic acid aptamer agent, the protein activity can be significantly inhibited at a lower concentration, and the inhibition time of the nucleic acid aptamer agent can be prolonged, as compared with the free nucleic acid aptamer agent. Therefore, the nucleic acid aptamer medicine is anchored on the surface of the cell membrane by utilizing the cell membrane anchoring sequence, the dosage of the nucleic acid aptamer medicine can be reduced, and the inhibition effect of the nucleic acid aptamer medicine is enhanced.
The main instruments used in this embodiment are:
Milli-Q INTEGARL pure water/ultrapure water integrated system (Millipore Co., U.S.A.); CARY ECLIPSE fluorescence spectrometer (Agilent Technologies, usa); small vertical electrophoresis tanks (Bio-Rad, inc., USA); chemiDocTM Touch gel imaging system (Bio-Rad, inc., USA); SH-1000UV-Vis Spectrophotometer (Corona Electric Co., japan); a1 confocal laser scanning microscope (Nikon Co., japan); ti-S inverted microscope (Nikon Co., japan); CHB 202 constant temperature metal bath (bosch technologies, hangzhou, china).
The main reagents used in this embodiment are:
The DNA sequences used in this experiment (Table 1) were all synthesized by Shanghai Biotechnology Co., ltd. And HPLC purified. Recombinant human HGF was purchased from PeproTech, U.S.A. anti-c-Met antibodies, anti-p-Met antibodies, anti-Akt antibodies, anti-p-Akt antibodies, anti-ERK 1/2 antibodies, anti-p-ERK 1/2 antibodies, and HRP-labeled secondary antibodies were purchased from CELL SIGNALING Technology Inc. MEM medium, dual antibody for cell culture and Fetal Bovine Serum (FBS) were purchased from Gibco company of the united states. RIPA cell lysate, protein concentration quantification kit, protease inhibitor and phosphatase inhibitor were purchased from shanghai bi yun biotechnology limited in china. DNase I was purchased from Thermo company in the united states. Phosphate Buffered Saline (PBS) was purchased from Gibco, inc. of the United states.
Example 1
The cell membrane anchored nucleic acid agent of this embodiment is assembled from a first anchoring nucleic acid sequence and a regulatory nucleic acid sequence comprising two functional sequences: a nucleic acid aptamer sequence and a second anchor nucleic acid sequence; wherein the nucleic acid aptamer sequence is a sequence shown in SEQ ID NO.1, the second anchor nucleic acid sequence and the first anchor nucleic acid sequence are completely complementary and hybridized to form a cell membrane anchor sequence with a double-chain structure, and the second anchor nucleic acid sequence and the first anchor nucleic acid sequence are both modified with a cell membrane anchor group. The second anchor nucleic acid sequence anneals to and complementarily hybridizes with the first anchor nucleic acid sequence to form a cell membrane anchor sequence.
Referring to the oligonucleotide sequences shown in Table 1 below, in example 1, a cell membrane receptor protein important in tumor cell activity, i.e., a mesenchymal transition factor (c-Met receptor protein), was selected as the target receptor protein, and a double-stranded complementary nucleic acid sequence of 18 bases was selected as the cell membrane anchor sequence. CH-b is the first anchor nucleic acid sequence modified with a cell membrane anchor group (in this example, a hydrophobic molecular cholesterol molecule), and b is shown in SEQ ID NO. 2. a is a nucleic acid aptamer sequence of a specific targeting c-Met receptor protein, CH-b is a second anchor nucleic acid sequence modified with cholesterol molecules, a second anchor nucleic acid sequence CH-b is added at the 5' end of the nucleic acid aptamer sequence a to form CH-ab, and a random base sequence T is arranged between the nucleic acid aptamer sequence a and the second anchor nucleic acid sequence b in the embodiment 1 in order to ensure that the configuration of the nucleic acid aptamer sequence is not influenced, wherein the sequence of b is shown in the following table 1, and the sequence of ab is shown in SEQ ID NO. 3. B in the second anchor nucleic acid sequence CH-b is fully complementary to b in the first anchor nucleic acid sequence CH-b. Both CH-b and CH-ab were measured at a molar concentration of 1:1, heating for 5min at 95 ℃ to anneal and hybridize with each other, forming a double-chain structure by CH-ab and CH-b, and slowly cooling to room temperature to form a cell membrane anchored nucleic acid drug 2 CH-ab. In another embodiment, a random base sequence disposed between the nucleic acid aptamer sequence a and the second anchor nucleic acid sequence b may be a or C or G; the number of other random base sequences may be not more than 5 bases as long as the configuration affecting the aptamer sequence is not affected.
Comparative example 1
The 2CH-ab: b in example 1 was replaced with the aptamer a of the unmodified cell membrane anchoring group ab: b and c-Met receptor protein, and the remaining experimental conditions were the same.
TABLE 1 oligonucleotide sequences used in example 1
In Table 1 above "-CH-" represents a cholesterol molecule.
Example 2
In this example 2, a target receptor protein was selected from the group consisting of a mesenchymal transition factor (c-Met receptor protein) which plays an important role in tumor cell activity, and a double-stranded complementary nucleic acid sequence of 24 bases was selected as a cell membrane anchor sequence. CH-d is a first anchor nucleic acid sequence modified with a cell membrane anchor group (in this example, a hydrophobic molecular cholesterol molecule), and d is as shown in SEQ ID No. 4; a is a nucleic acid aptamer specifically targeting c-Met receptor protein, CH-d is a second anchor nucleic acid sequence modified with cholesterol molecules, the second anchor nucleic acid sequence CH-d is added at the tail end of nucleic acid aptamer a of c-Met receptor protein to form CH-ad, and in order to ensure that the configuration of the nucleic acid aptamer sequence is not affected, a random base sequence T is arranged between the nucleic acid aptamer sequence a and the second anchor nucleic acid sequence d in the embodiment 2, wherein the sequence of ad is shown as SEQ ID NO.5, and the sequences of d and d are completely complementary. The corresponding nucleic acid sequences were prepared as in example 1, and the sequences of CH-ad and CH-d in this example 2 are shown in Table 2.
Comparative example 2
Instead of the 2CH-ad of example 2, the d of the unmodified cell membrane anchoring group was replaced, and the remaining experimental conditions were the same.
TABLE 2 oligonucleotide sequences used in example 2 and comparative example 2
In Table 2 above "-CH-" represents a cholesterol molecule.
Related experimental procedures of example 1, comparative example 1, example 2, comparative example 2
The nucleic acid probes in example 1, comparative example 1, example 2, and comparative example 2 were subjected to the following experiments:
A. Anchor stability optimization of cell membrane anchoring sequences
Optimization of the number and position of cholesterol molecule modifications: after incubation of 200nM of the different cell membrane anchoring sequences (2 CH-b: b, 2CH-c: c, CH-b: b, CH-b and CH-c) with HeLa cells for 10min, excess probe was washed off with PBS and confocal fluorescence imaging was performed. Cells were then incubated with medium containing 10% fetal bovine serum for 30 minutes, washed 3 times with PBS, and confocal fluorescence imaging was performed again.
B. Anchoring effect and anchoring directionality verification
200NM 2CH-ab: b was incubated with HeLa cells for 10 min, washed 3 times with PBS and confocal fluorescence imaged to verify its anchoring effect. To verify its anchoring directionality, DNase I (0.5U/. Mu.l) was then added for 5 min incubation with cells, and confocal fluorescence imaging was performed again after 3 washes in PBS.
C. nucleic acid aptamer competition experiments
10 Mu M CH-ab and 10 mu M CH-b are mixed, annealed for 5 minutes at 95 ℃, cooled to room temperature for more than 2 hours and stored at 4 ℃. To characterize the stability of aptamer binding, first confocal fluorescence imaging was performed by incubating HeLa cells with 400nM of Cy3 fluorophore-labeled a (Cy 3-Free a) or Cy3 fluorophore-labeled 2CH-ab: b (Cy 3-2CH-ab: b) for 10min, washing 3 times with PBS. Then, cells were incubated with 400nM of Cy5 fluorophore-labeled a (Cy 5-Free a) for 15 min, washed 3 times with PBS and then imaged a second time.
D. immunoblotting experiments
After culturing DU145 cells in 6-well plates (3×10 5 cells per well) for 24 hours, the culture was replaced with MEM medium containing 0.5% bsa and starved for 24 hours, and experiments were performed:
(1) Performance investigation of cell membrane anchored nucleic acid drug enhancement of inhibitory protein activity effect: DU145 cells were incubated with nucleic acid probes (2 CH-ab: b, or a) at different concentrations for 15min, then 20ng/mL HGF was added to incubate the cells for 30 min, after washing the cells 3 times with cold PBS, the cells were lysed with RIPA lysis buffer with protease inhibitor (1X) and phosphatase inhibitor (1X) added, protein was extracted and the concentration was determined, and immunoblotting experiments were performed.
To verify the versatility of this strategy, 30nM of the different nucleic acid probes (2 CH-ad: d or 2CH-d: d) were incubated for 15min, 20ng/mL HGF was added to incubate the cells for 30min, the cells were washed 3 times with cold PBS, lysed with RIPA lysis buffer with protease inhibitor (1X) and phosphatase inhibitor (1X), protein was extracted and the concentration was determined before performing an immunoblotting experiment.
(2) Performance investigation of cell membrane anchored nucleic acid drug to extend inhibition time: after 250nM2CH-ab: b or ab: b was added to DU145 cells, incubation was performed for 24 hours in the cell incubator. After further incubation with HGF at 20ng/mL for 30min, the cells were washed 3 times with cold PBS, the cells were lysed to extract the protein, and immunoblotting experiments were performed after measuring the protein concentration. Cells incubated for 30min with addition of only 20ng/mL HGF served as positive control.
E. Analysis of cell migration Capacity
DU145 cells were seeded in 12-well plates to confluence throughout the plates. Experiments were performed after starving cells for 24 hours by replacing the complete medium with MEM medium containing 0.5% bsa. Scratches were made in the center of the well plate using a sterile 200 μl gun head and the scratch width was recorded under an inverted microscope. Cell migration experiments were divided into four groups. A first group: blank control group; second group: only 20ng/mL HGF was added as a positive control group; third group: 30nm ab: b; fourth group: 30nM 2CH-ab, b. After the third and fourth groups were incubated for 15 minutes with nucleic acid probes, 20ng/mL HGF was added for incubation. After each group of cells was placed in an incubator for continuous culture for 24 hours, the scratch width was recorded under an inverted microscope. Migration distance of cells within 24 hours was quantified using Image J software.
(II) results of related experiments
A. Anchor stability optimization of cell membrane anchoring sequences
First, to achieve the best anchoring effect, we optimized the number and position of cholesterol molecules modified on the anchoring nucleic acid sequence, and constructed 5 cell membrane anchoring sequences, including 2ch_b, 2ch_c, ch_b, and ch_c, respectively, to examine their stability on cell membranes. Wherein, b is a double-chain cell membrane anchoring sequence, and a cholesterol molecule is modified at the middle position of two anchoring nucleic acid sequences (a first anchoring nucleic acid sequence and a second anchoring nucleic acid sequence); 2CH-c is a double-stranded cell membrane anchor sequence, modified with a cholesterol molecule at each of the 3 'end of one anchor nucleic acid sequence and the 5' end of the other anchor nucleic acid sequence; CH-b is a double-stranded cell membrane anchor sequence, wherein a cholesterol molecule is modified in the middle of one of the anchor nucleic acid sequences; CH-b is a single-stranded anchor nucleic acid sequence, and a cholesterol molecule is modified at the middle position of the sequence; CH-c is a single stranded anchor nucleic acid sequence, modified at the 5' end of the sequence with a cholesterol molecule. We labeled Cy3 fluorescence on both of these anchor nucleic acid sequences. As shown in the confocal images of FIGS. 2A-2E, these cell membrane anchoring nucleic acid sequences can successfully anchor to the cell membrane surface. In the presence of 10% fetal bovine serum, double-stranded nucleic acid sequences (i.e., 2CH-b: b) that modify two cholesterol molecules at a position intermediate the cell membrane anchoring nucleic acid sequences exhibit more stable cell membrane anchoring properties. By increasing the number of cell membrane anchoring molecules (i.e. cell membrane anchoring groups) a better anchoring stability can be obtained compared to CH-b: b. Compared with 2CH-c, when the cell membrane anchoring molecule is modified at the middle position of the double-chain anchoring nucleic acid sequence, the cell membrane anchoring nucleic acid sequence with double-chain structure can be inserted into the deeper position of the cell membrane, so that the anchoring stability is improved. Thus, 2CH-b: b was chosen as the cell membrane anchoring sequence for subsequent experiments.
From the above experimental results, it can be seen that: (1) Two cholesterol molecules are respectively modified at the middle positions of the first anchoring nucleic acid sequence and the second anchoring nucleic acid sequence, and the cholesterol molecules are positioned at symmetrical positions of double chains after the hybridization of the first anchoring nucleic acid sequence and the second anchoring nucleic acid sequence; (3) Cholesterol is one of the hydrophobic molecules, which is inserted into the phospholipid layer of the cell membrane by its hydrophobicity, and so on to other hydrophobic molecules such as tocopherol molecules and diacyl liposomes, which also have better effects in modifying the middle position of the double-stranded anchored nucleic acid sequence.
B. construction and characterization of cell membrane anchored nucleic acid drugs
In example 1, comparative example 1, example 2 and comparative example 2, the c-Met receptor protein was selected as the target receptor protein model, and a nucleic acid aptamer targeting the c-Met receptor protein, namely a, was selected to verify our design. The c-Met receptor protein is an important drug target, and its overactivation leads to migration and invasion of tumor cells. The second anchor nucleic acid sequence, CH-ab, is extended at the end of aptamer a and hybridized to the first anchor nucleic acid sequence CH-b to form 2CH-ab: b. We first validated the hybridization of the nucleic acid drug in PBS buffer. As shown in FIG. 3, from left to right, lane 1 is the CH-ab band, lane 2 is the CH-b band, and since CH-ab hybridizes to CH-b, lane 3 shows a new band at a higher position, demonstrating successful assembly of 2CH-ab:b, demonstrating that modification of cholesterol molecule does not affect CH-ab to CH-b hybridization. Next, as shown in fig. 4A, we labeled Cy3 fluorescence at the 3 'end of probe CH-ab and Cy5 fluorescence at the 5' end of probe CH-b. After incubating the assembled 2CH-ab: b with HeLa cells for ten minutes, fluorescence of Cy3 and Cy5 was observed on the cell membrane at the same time, demonstrating that the 2CH-ab: b was successfully anchored to the cell membrane surface of HeLa cells.
In example 2, to verify the versatility of the method, we designed a24 base length first and second anchor nucleic acid sequence, designed the nucleic acid drug 2 CH-ad. As shown in FIG. 4B, 2CH-ad was successfully anchored to the cell membrane surface of HeLa cells.
The ability of nucleic acid aptamer drugs to recognize and bind to target proteins is critical for their inhibitory effect, and thus requires that the aptamer be located outside the cell membrane. To examine the directionality of anchoring of 2CH-ab b on cell membranes, we labeled Cy3 fluorescence at the 3 'end of probe CH-ab and Cy5 fluorescence at the 3' end of probe CH-b. As shown in FIG. 5, the assembled probe 2CH-ab: b was anchored to HeLa cell membrane, and a bright circle of fluorescence of Cy3 and Cy5 could be observed simultaneously. DNase I is an endonuclease capable of hydrolyzing single-stranded or double-stranded DNA. Since DNase I cannot cross cell membranes, we used it to examine the directionality of 2CH-ab: b anchorage. Cy3 fluorescence on the cell membrane after DNase I treatment was significantly reduced, while Cy5 fluorescence remained essentially unchanged, indicating that aptamer a was located outside the cell membrane. Next, we examined the stability of binding of 2CH-ab: b to the target c-Met receptor protein by a nucleic acid aptamer competition experiment. As shown in FIG. 6A, almost no Cy5 fluorescence signal was present on the Cy3-2CH-ab: b anchored cell membrane. However, a distinct Cy5 fluorescent signal appeared on the cell membrane incubated with Cy3-Free a, indicating binding of Cy5-Free a (FIG. 6B). It was thus verified that cell membrane anchored 2CH-ab: b is able to bind more stably to the target c-Met receptor protein than the free aptamer a.
C. cell membrane anchored nucleic acid drugs enhancing inhibitor protein activity and cellular function
In example 1, comparative example 1, example 2 and comparative example 2, c-Met receptor protein was selected as a target receptor protein model, and whether or not a cell membrane-anchored nucleic acid drug could increase inhibition of c-Met receptor phosphorylation by HGF was examined. We also compared the effect of probe 2CH-ab: b, ab: b on c-Met receptor activity inhibition by free a. As shown in fig. 7A and fig. 7B, from the results of immunoblot analysis experiments, the inhibition capability of 2CH-ab: B was significantly better than ab: B, and the protein expression of phosphorylated c-Met (p-Met) could be inhibited at a lower concentration. Meanwhile, ab-B shows similar inhibition ability with free a (shown in fig. 7B and fig. 8), and further verifies that 2 CH-ab-B which can be anchored on cell membranes can obviously enhance the inhibition effect of the nucleic acid aptamer medicine, and can reduce the dosage of the nucleic acid aptamer medicine.
In example 2, to verify the versatility of the method, we designed a 24 base length first and second anchor nucleic acid sequence as another set of cell membrane anchor sequences, followed by the design of the nucleic acid drug 2 CH-ad. Protein expression of p-Met was barely detectable in cells treated with 30nM 2CH-ad, whereas cells treated with the same concentration of free ad showed similar levels of p-Met expression as in the positive control group treated with HGF alone, and failed to exert a significant inhibitory effect. Meanwhile, the cell membrane anchoring sequence (2 CH-d: d) does not affect the activity of the c-Met receptor protein. Experimental results prove that the cell membrane anchored nucleic acid drug 2CH-ad is capable of inhibiting the activity of c-Met receptor protein more effectively (see the result of figure 9), and the universality of the strategy is demonstrated.
As can be seen from the comparison of example 1 and example 2, the first anchor nucleic acid sequence and the second anchor nucleic acid sequence are not limited to the specific sequences listed in the above-mentioned examples 1 and 2, as long as the first anchor nucleic acid sequence and the second anchor nucleic acid sequence satisfy the cell membrane anchor sequences forming a double-stranded structure of 18-24bp stable hybridization, and the first anchor nucleic acid sequence and the second anchor nucleic acid sequence are both modified with the cell membrane anchor groups. Because the length of the aptamer sequence in the scheme is 50 bases, the length of the cell membrane anchoring sequence is not too long, and if the length is too long, on the one hand, the difficulty of modifying cholesterol molecules is increased, the DNA raw materials are wasted, and the cost of synthesizing DNA is increased; on the other hand, too long a cell membrane anchor sequence may affect the function of the aptamer sequence. The experimental results of example 1 and example 2 show that, by allowing the nucleic acid aptamer to carry a membrane anchoring sequence with a double-stranded structure of a proper length (18-24 bp) on the nucleic acid aptamer sequence and binding the membrane anchoring nucleic acid sequence with a membrane anchoring group on the membrane anchoring sequence, the nucleic acid aptamer can be enriched on the surface of the membrane, meanwhile, the binding stability of the nucleic acid aptamer with a membrane receptor protein is improved, the activity of the c-Met receptor protein can be obviously inhibited at a lower concentration, and the inhibition effect of the nucleic acid aptamer drug is enhanced.
Activation of c-Met receptor proteins is closely related to cell migration behavior. Therefore, we examined the effect of 2CH-ab: b on the regulation of cell behavior by cell scratch experiments. Consistent with the expected effect, as shown in fig. 10, the rate of healing scratches was slowed by applying 2CH-ab: b incubation, showing better ability to inhibit cell migration behavior.
D. Cell membrane anchored nucleic acid agents for prolonged inhibition
Although both probes 2CH-ab: b and ab: b showed good inhibition in a short period of time (15 minutes) of 250nM (FIG. 7). As shown in FIG. 11, only 2CH-ab: b was able to resist HGF stimulus-mediated activation of c-Met receptor after 24 hours incubation with cells, and still maintained good inhibitory effect. Whereas ab b had substantially no inhibition after 24 hours incubation with cells, the p-Met protein expression levels had been comparable to the positive control group treated with HGF alone. Experimental results indicate the feasibility of 2CH-ab: b in long-acting inhibition of protein activity.
The analysis of the experimental results shows that the cell membrane anchoring sequence with the double-chain structure constructed by the invention can enrich more aptamer medicines on the surface of the cell membrane and increase the binding stability (see the result of the attached drawing 6), so that a better inhibition effect can be realized only by smaller dosage (see the results of the attached drawing 7 to the attached drawing 10), and meanwhile, the cell membrane anchoring sequence can slow down the process of endocytosis of the aptamer medicines by cells, and the inhibition time is prolonged aiming at targets on the cell membrane of the c-Met receptor protein (see the result of the attached drawing 11)
The cell membrane anchored nucleic acid medicine constructed by the invention can obviously improve the combination stability of the nucleic acid aptamer medicine and the target protein, can obviously inhibit the activity of c-Met receptor protein at a lower concentration, and greatly enhances the inhibition effect of the nucleic acid aptamer medicine. In addition, the strategy can effectively prolong the acting time of the aptamer medicine on the surface of a cell membrane and reduce the loss of medicine effect caused by endocytosis. The designed cell membrane anchored nucleic acid medicine is simple and controllable, provides a new platform for developing high-efficiency nucleic acid medicine, and further expands the application prospect in biomedicine.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the scope of protection thereof, and although the present application has been described in detail with reference to the above embodiments, it should be understood by those of ordinary skill in the art that: various alterations, modifications, and equivalents may be suggested to the detailed description as would occur to one skilled in the art after reading this disclosure, and are intended to be within the scope of the appended claims.
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Claims (4)
1. A cell membrane anchored nucleic acid agent, comprising a first anchoring nucleic acid sequence and a regulatory nucleic acid sequence, wherein the regulatory nucleic acid sequence comprises two functional sequences: a nucleic acid aptamer sequence and a second anchor nucleic acid sequence; the 5' end of the nucleic acid aptamer sequence is connected with the second anchoring nucleic acid sequence, the nucleic acid aptamer sequence specifically targets cell membrane receptor protein, the nucleic acid aptamer sequence is shown in SEQ ID NO.1, the second anchoring nucleic acid sequence and the first anchoring nucleic acid sequence are completely complementary and hybridized to form a cell membrane anchoring sequence with a double-chain structure, and one or two cell membrane anchoring groups are modified on the cell membrane anchoring sequence;
1-5 random base sequences are arranged between the nucleic acid aptamer sequence and the second anchor nucleic acid sequence; the cell membrane anchoring group is one of hydrophobic molecules including cholesterol molecules, tocopherol molecules and diacyl liposomes;
Complementary hybridization of the first anchor nucleic acid sequence and the second anchor nucleic acid sequence forms a double-stranded structure with a length of 18-24 bp;
The cell membrane receptor protein is a mesenchymal epidermal transforming factor which can be activated by receptor dimerization mediated by a ligand hepatocyte growth factor.
2. The nucleic acid drug of claim 1, wherein both of said cell membrane anchoring groups are modified at intermediate positions of said first anchoring nucleic acid sequence and said second anchoring nucleic acid sequence; the intermediate position means: the first anchoring nucleic acid sequence and the second anchoring nucleic acid sequence are n bases, and when n is even, two cell membrane anchoring groups are respectively modified on DNA skeletons between the n/2 th and (n/2) +1 th bases of the first anchoring nucleic acid sequence and the second anchoring nucleic acid sequence; when n is an odd number, two of the cell membrane anchoring groups are modified on the DNA backbone intermediate the (n/2) -0.5 and (n/2) +0.5 bases of the first and second anchoring nucleic acid sequences, respectively.
3. The nucleic acid drug of claim 1, wherein the nucleic acid aptamer sequence overlaps or is identical to the binding site of the cell membrane receptor protein and the ligand partially overlaps or is identical to the binding site of the cell membrane receptor protein.
4. A method for preparing a nucleic acid drug according to any one of claims 1-3, comprising the steps of:
S1, synthesizing a first anchor nucleic acid sequence;
s2, synthesizing a regulatory nucleic acid sequence;
s3, mixing the two nucleic acid sequences in the steps S1 to S2 according to the molar concentration of 1:1, heating at 95 ℃ for 5 minutes for annealing and hybridizing, and slowly cooling to room temperature.
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| WO1988005077A1 (en) * | 1986-12-29 | 1988-07-14 | Battelle Memorial Institute | A method of modifiying the metabolism of eukaryotic cells upon incorporation therein of foreign sequences of nucleic acids by means of an internalizing vector |
| WO2013177046A1 (en) * | 2012-05-21 | 2013-11-28 | Solulink, Inc. | Methods and/or use of oligonucleotide conjugates for suppressing background due to cross-hybridization |
| CN109402127A (en) * | 2018-09-29 | 2019-03-01 | 复旦大学附属眼耳鼻喉科医院 | One group of high affinity nucleic acid aptamers and its application specifically bound with Connective Tissue Growth Factor |
| CN110283826A (en) * | 2019-07-03 | 2019-09-27 | 福州大学 | A kind of bispecific aptamer, derivative, preparation method and applications |
| CN112852822A (en) * | 2021-01-19 | 2021-05-28 | 中南大学 | Na based on aptamer+、K+Simultaneous detection fluorescent nano probe and preparation method and application thereof |
Family Cites Families (2)
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| AU2002252885A1 (en) * | 2001-04-24 | 2002-11-05 | Mcgill University | A 150 kda tgf-beta 1 accessory receptor acting as a negative modulator of tgf-beta signaling |
| US20170335314A1 (en) * | 2016-05-23 | 2017-11-23 | Tribiotica Llc | Methods For Using Nucleic Acid Aptamers For Directed Templated Assembly |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1988005077A1 (en) * | 1986-12-29 | 1988-07-14 | Battelle Memorial Institute | A method of modifiying the metabolism of eukaryotic cells upon incorporation therein of foreign sequences of nucleic acids by means of an internalizing vector |
| WO2013177046A1 (en) * | 2012-05-21 | 2013-11-28 | Solulink, Inc. | Methods and/or use of oligonucleotide conjugates for suppressing background due to cross-hybridization |
| CN109402127A (en) * | 2018-09-29 | 2019-03-01 | 复旦大学附属眼耳鼻喉科医院 | One group of high affinity nucleic acid aptamers and its application specifically bound with Connective Tissue Growth Factor |
| CN110283826A (en) * | 2019-07-03 | 2019-09-27 | 福州大学 | A kind of bispecific aptamer, derivative, preparation method and applications |
| CN112852822A (en) * | 2021-01-19 | 2021-05-28 | 中南大学 | Na based on aptamer+、K+Simultaneous detection fluorescent nano probe and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 核酸适配体技术及其在肿瘤诊断和治疗中的应用;孙红光;张金三;吴建波;祖幼立;朱迅;;药学进展(08);全文 * |
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